Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells

Abstract

The transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate subsites are required for the preferred substrates, Cl- and HCO3-, to account for tight binding in the transition state (Kdiss congruent to 1 microM). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unusual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and "tunneling," which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.

Transport domain of the erythrocyte anion exchange protein

“…These conformational changes induced by bulky substrate analogs such as PLP, might be of relevance for understanding the accommodation of a large variety of anions by the transport site and the dynamics of translocation of the transportable anions. The conformational flexibility associated with substrateinduced changes in the transport domain of AEP, might be an intrinsic property of the anion exchange mechanism, which is consistent with many features of the anion exchange system and with the recently proposed (transition-state) theory which calls for a carrier conformational change in the process of substrate-binding stabilization [28,29].…”

A Conserved Glutamate Is Responsible for Ion Selectivity and pH Dependence of the Mammalian Anion Exchangers AE1 and AE2

“…Size alone cannot account for the observed differences in maximum transport rates, as the distribution in maximal transport rates is not well correlated with molecular geometry. These observations led Krupka to propose that stabilization of the transition state in carrier reorientation is the physical basis for anion selectivity (Krupka, 1989). In this context, our data suggest that the WRK glutamate contributes critically to this stabilization.…”

Membrane cation and anion transport activities in erythrocytes of hereditary spherocytosis: Effects of different membrane protein defects

“…According to the working model of band 3 proposed by Krupka [37,38], the altered function of the anion transporter may be interpreted as a decrease in the activation energy or as a structural change in the protein during the translocation step, or as an increase in stability of the transition state intermediate. It is possible that band 4.2, through its interaction with the band 3 cytoplasmatic domain, is keeping the transmembrane domain in a particular conformation that is not the most favorable for maximal anion transport [37,38]. A similar mechanism of modulation occurs when hemoglobin binds to the cytoplasmatic domain of band 3: binding of hemoglobin determines kinetic changes in anion transport activity, suggesting that conformational changes in the cytoplasmatic domain influence the transmembrane domain and modulate the function of the anion transporter.…”